Mechanisms of homeostatic plasticity ensure that cells maintain spiking activity levels within a physiologically relevant window. This form of plasticity prevents cells from falling silent or from becoming hyperexcitable. The homeostatic control of spiking activity is critically important to nervous system function, as demonstrated by the severity of conditions like seizure, where such homeostasis is not achieved. Homeostatic plasticity is thought to underlie the robustness of network behavior, and allow for the recovery of behaviors following perturbations. The last 15 years have seen a dramatic rise in studies of homeostatic plasticity, which have now demonstrated multiple mechanisms, which contribute to the resilience of spiking activity following perturbations. By far the most studied mechanism of homeostatic plasticity has been referred to as synaptic scaling. When spiking activity in cultured neurons is blocked for days, all of a cell's excitatory synapses strengthen, or scale up. Current thinking in the field suggests that synaptic scaling is triggered by reduced spiking activity, whic then acts to recover normal spiking levels. In this proposal we challenge this very basic, but largely untested assumption. We hypothesize that synaptic scaling is triggered by reductions in synaptic transmission at individual synapses to homeostatically maintain synaptic transmission, rather than as a means to homeostatically regulate a cells spiking activity. We will test this hypothesis using a combination of optogenetics and multi-electrode array recordings of cultured neurons. We will ask in Aim 1 excitatory upscaling is triggered by reduced glutamatergic transmission? Then we will ask in Aim 2 if excitatory downscaling is triggered by increased glutamatergic transmission? In Aim 3 we will determine if alterations in spiking or transmission trigger GABAergic scaling. If the results favor neurotransmission as the trigger for synaptic scaling, this could provide a transformative shift in the focus of synaptic scaling away from the activity-centric perception that is currently pervasive. This proposal will use a combination of new techniques that allow the assessment and control of spiking activity levels to ask fundamentally important questions about homeostatic plasticity. The results will have significant implications for understanding how circuits change following injury and disease.